Single Mucosal, but Not Parenteral, with Recombinant Adenoviral-Based Provides Potent Protection from Pulmonary This information is current as of October 1, 2021. Jun Wang, Lisa Thorson, Richard W. Stokes, Michael Santosuosso, Kris Huygen, Anna Zganiacz, Mary Hitt and Zhou Xing J Immunol 2004; 173:6357-6365; ;

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2004 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Single Mucosal, but Not Parenteral, Immunization with Recombinant Adenoviral-Based Vaccine Provides Potent Protection from Pulmonary Tuberculosis1

Jun Wang,* Lisa Thorson,† Richard W. Stokes,† Michael Santosuosso,* Kris Huygen,‡ Anna Zganiacz,* Mary Hitt,* and Zhou Xing2*

Bacillus Calmette-Gue´rin (BCG) vaccine has failed to control the global tuberculosis (TB) epidemic, and there is a lack of safe and effective mucosal capable of potent protection against pulmonary TB. A recombinant replication-deficient adenoviral- based vaccine expressing an immunogenic tuberculosis Ag Ag85A (AdAg85A) was engineered and evaluated for its potential to be used as a respiratory mucosal TB vaccine in a murine model of pulmonary TB. A single intranasal, but not i.m., immunization with AdAg85A provided potent protection against airway Mycobacterium tuberculosis challenge at an improved level over that by cutaneous BCG . Systemic priming with an Ag85A DNA vaccine and mucosal boosting with Downloaded from AdAg85A conferred a further enhanced immune protection which was remarkably better than BCG vaccination. Such superior protection triggered by AdAg85 mucosal immunization was correlated with much greater retention of Ag-specific T cells, par- ticularly CD4 T cells, in the lung and was shown to be mediated by both CD4 and CD8 T cells. Thus, adenoviral TB vaccine represents a promising novel vaccine platform capable of potent mucosal immune protection against TB. Our study also lends strong evidence that respiratory mucosal vaccination is critically advantageous over systemic routes of vaccination against TB. The Journal of Immunology, 2004, 173: 6357–6365. http://www.jimmunol.org/

uberculosis (TB)3 is a chronic respiratory infectious dis- Increasing evidence suggests that vaccination at the mucosal site ease that has afflicted humans for thousands of years. For is superior to vaccination at other sites in eliciting protection from T Ͼ80 years, the bacillus Calmette-Gue´rin (BCG) vaccine mucosal infectious diseases (6). This is partially explained by the has been the only licensed TB vaccine given to humans covering observation that memory T and B cells generated upon mucosal 86% of the world population in 2001 (1). However, despite the use vaccination acquire mucosa-homing receptors and preferentially of BCG, TB remains a global epidemic with one-third of the world accumulate at the mucosal site of induction (7, 8). Thus, it is be-

population being infected and an annual rate of 8 million new lieved that greater immune protection may be achieved if TB vac- by guest on October 1, 2021 cases and 2–2.5 million deaths (2). Regardless of its protection cine is given mucosally via the respiratory tract (9, 10). However, from severe forms of childhood TB, BCG fails to confer protection few mucosally delivered TB vaccines, except replicating myco- from adult TB (3, 4). Furthermore, BCG vaccine may cause severe bacteria, could successfully trigger protective immune responses in complications in immunocompromised hosts (5). Thus, there is an the lung (11–14). Detrimental immunopathology that may be urgent need for developing safe and effective TB vaccines that are caused by direct application of live mycobacterial vaccines at the able to confer potent protection at the respiratory mucosa. mucosal site, especially at the respiratory tract, is of particular safety concern (5, 9). Furthermore, although promising, recombi- nant plasmid DNA or protein/subunit vaccines, when given par- enterally, are unable to elicit superior protection over BCG vaccine *Department of Pathology and Molecular Medicine and Division of Infectious Dis- (15–17) and when given mucosally cannot lead to effective im- eases, Centre for Gene Therapeutics, McMaster University, Hamilton, Ontario, Can- ada; †Department of Paediatrics, British Columbia Research Institute for Children and mune activation (17–20). Therefore, the development of safe and Women’s Health, University of British Columbia and British Columbia Children’s efficacious respiratory mucosal TB vaccines has remained a major Hospital, Vancouver, British Columbia, Canada; and ‡ of Brussels, Brussels, challenge to TB vaccinologists. Received for publication May 28, 2004. Accepted for publication September Adenovirus with a natural tropism to the respiratory epithelium 10, 2004. may cause mild cold symptoms of the upper airways in humans. In The costs of publication of this article were defrayed in part by the payment of page this regard, a live wild-type has been given charges. This article must therefore be hereby marked advertisement in accordance mucosally to military recruits in North America to prevent adeno- with 18 U.S.C. Section 1734 solely to indicate this fact. viral (21). Thus, due to ample safety data and its low 1 This study was supported by grants from the World Health Organization, Sequella Global Tuberculosis Foundation, Ontario Government, and Canadian Institutes for , tropism to mucosal epithelial cells, and superior gene Health Research (to Z.X.), and the Canadian Bacterial Diseases Network/National transfer efficiency, genetically engineered replication-deficient ad- Centres of Excellence (to R.W.S.). R.W.S. is the recipient of a British Columbia Research Institute for Children’s and Women’s Health Investigatorship Award. enovirus has been widely used for the purpose of gene transfer in vivo (22). In addition to its applications in gene replacement ther- 2 Address correspondence and reprint requests to Dr. Zhou Xing, Room 4012-MDCL, Department of Pathology and Molecular Medicine, McMaster University, 1200 Main apy, there is an increasing interest in using adenoviral vector to Street West, Hamilton, Ontario, Canada L8N 3Z5. E-mail address: [email protected] deliver transgenes encoding immunogenic microbial Ags for vac- 3 Abbreviations used in this paper: TB, tuberculosis; BCG, bacillus Calmette-Gue´rin; cination against infectious diseases such as AIDS, herpes infec- Ad, adenoviral; t-PA, tissue plasminogen activator signal peptide; CFP, culture filtrate protein; ICCS, intracellular cytokine staining; i.n., intranasal; M.tb, Mycobacterium tion, and malaria (23–25). Adenoviral vector when used for the tuberculosis. purpose of vaccination, as opposed to gene replacement therapy,

Copyright © 2004 by The American Association of Immunologists, Inc. 0022-1767/04/$02.00 6358 VIRAL-MEDIATED MUCOSAL IMMUNITY AGAINST TB possesses type 1 immune adjuvant properties and gives rise to promoter and a SV40 poly(A) signal in orientation. This recombinant plas- self-limited but prolonged high levels of Ag release, which are mid (pJW24) was cotransfected into 293 cells along with a rescuing vector believed to favor long-term memory immune responses against pBHG10 (28) (Fig. 1a) that contained the entire type 5 human adenovirus genomic DNA sequences, except the E1, E3, and packaging regions. Re- infection (26). However, TB vaccination by using adenoviral vec- combinant replication-deficient adenoviral vector AdAg85A was then res- tors has not been explored. Given the natural tropism of adenovirus cued by homologous recombination. Similarly, the t-PA/Ag85A segment to the respiratory epithelium, recombinant replication-deficient ad- was inserted into the multicloning site of plasmid vector pCDNA3.1 to enoviral vector engineered to express selected immunogenic My- obtain plasmid DNA vaccine DNAAg85A. The correctness of AdAg85A or DNAAg85A was verified by Northern and Southern blot hybridizations. cobacterium tuberculosis (M.tb) Ags is an ideal candidate of re- AdAg85A was amplified, purified, and titrated according to the protocols spiratory mucosal TB vaccine. previously described (29). The secretion of Ag85A protein by AdAg85A- In the present study, we have developed a recombinant replica- infected mammalian cells was verified by Western immunoblotting using a tion-deficient adenoviral vector expressing a single immunogenic mAb (clone TD-17) and HRP-conjugated anti-mouse IgG. M.tb Ag, Ag85A (AdAg85A). We demonstrate here that a single Immunization with AdAg85A, DNAAg85A, or BCG and respiratory mucosal vaccination with AdAg85A induces the acti- depletion of CD4 and CD8 T cells vation of both Ag-specific CD4 and CD8 T cells that are different both geographically and qualitatively from those induced by par- Six- to 8-wk-old female BALB/c mice were purchased from Harlan Lab- oratory (Indianapolis, IN) and maintained under specific -free enteral vaccination, and it results in potent protection against re- conditions at the McMaster University Central Animal Facility or the An- spiratory M.tb challenge. Furthermore, a heterologous prime-boost imal Facility at the British Columbia Research Institute for Children’s and regimen involving systemic priming with a plasmid DNA vaccine Women’s Health, University of British Columbia. For intranasal (i.n.) de- and respiratory mucosal boosting with AdAg85A triggers an even livery of AdAg85A, 5 ϫ 107 PFU of AdAg85A or Addl70-3 were diluted ␮ Downloaded from further enhanced protection which is remarkably superior to that with PBS to a total volume of 30 l and delivered into mouse airways in two aliquots with a fine pipette tip. For i.m. delivery, 5 ϫ 107 PFU of by cutaneous BCG vaccination. AdAg85A or Addl70-3 was diluted with PBS to a total volume of 100 ␮l and injected into both hind legs (two separate injections of 50 ␮l/leg) (28). Materials and Methods BCG (Connaught or Pasteur strain) at the dose of ϫ 5 ␮ Construction and characterization of recombinant 5 10 CFU/mouse was diluted with PBS in a total volume of 100 l and s.c. injected into mice around each hind leg (14). For DNA immunization, replication-deficient adenoviral vaccine (AdAg85A) and plasmid 50 ␮g of DNAAg85A was diluted in 50 ␮l of PBS and injected i.m into a http://www.jimmunol.org/ DNA vaccine (DNAAg85A) hind leg of the mice. In some experiments, vaccinated mice were depleted of CD4 or CD8 T cells or both, 2 days before M.tb challenge, by four Ag85A cDNA was amplified by PCR from M.tb genomic DNA (H Rv). 37 repeated weekly i.p. injections of monoclonal anti-CD4 (GK1.5) and -CD8 The Ag85A segment without endogenous signal peptide was first amplified (2.43) T cell Abs. by primers (sense primer, GATGTTGTGTCTGTTCGGAG; antisense primer, GATGAGGGAAGCAAGAATGC) and was reamplified with a Lymphocyte isolation and in vitro Ag stimulation nested pair of primers that contained restriction sites of BglII and EcoRI at the 5Ј end of sense and antisense primers. A 900-bp BglII/EcoRI fragment At various times postvaccination, spleens and popliteal lymph nodes or of Ag85A was isolated and ligated into the plasmid vector containing se- mediastinal lymph nodes draining the site of immunization were removed quences coding for the human tissue plasminogen activator signal peptide and pooled for each group. Cells were isolated and cultured as previously (t-PA) in such a way that the Ag85A cDNA and t-PA signal peptide se- described (30, 31). Approximately 0.5 ϫ 106 cells were seeded into 96- by guest on October 1, 2021 quences were in-frame and that protein translation would start at t-PA. The well plates in RPMI 1640 medium containing 10% FCS, 100 U/ml peni- t-PA/Ag85A segment was then inserted into the multicloning site of a cillin, 100 g/ml streptomycin, and 2 mmol/L of L-glutamine at 37°C with shuttle plasmid vector, pVDB3 (27), in a position between a murine CMV or without Ag stimulation. The Ags used to stimulate cells included M.tb

FIGURE 1. Construction of AdAg85A (a). A shuttle plasmid vector pJW24 was designed as shown in the diagram and constructed by multiple subcloning steps as detailed in Mate- rials and Methods. The presence of adenoviral sequences in pJW24 allows homologous re- combination with adenoviral rescuing vector pBHG10 to form recombinant replication-de- ficient adenovirus AdAg85A, which has the entire expression cassette inserted into the E1 region of the adenoviral genome. Character- ization of AdAg85A by Northern blot hybrid- ization (b) and Western blot (c). Total cellular RNA and culture supernatant were prepared from A549 cells infected with AdAg85A or control vector Addl70-3 and subjected to Northern blot hybridization with a 32P-labeled Ag85A cDNA probe and Western blotting with an anti-Ag85A TD-17 mAb, respectively. The Journal of Immunology 6359

culture filtrate protein (CFP; 8 ␮g/ml) or purified M.tb Ag85 complex detected in the culture supernatant from A549 cells infected with proteins (8 ␮g/ml). The culture supernatants were collected at 72 h. Stim- AdAg85A but not Addl70-3 (Fig. 1c). ulation with irrelevant Ags was used as control and resulted in only back- ground levels of cytokine production (not graphed in the figures for the Ag-specific type 1 immune activation by AdAg85A in vivo purpose of simplicity). We next examined whether AdAg85A was able to elicit Ag-spe- Intracellular cytokine staining (ICCS), flow cytometry analysis cific immune responses in vivo and, if so, what was the nature of (FACS), and ELISPOT assay such Ag85A-specific immunity. BALB/c mice were injected with The frequency of Ag-specific, IFN-␥-releasing T cells was examined by various doses of AdAg85A or control virus Addl70-3 via the i.m. ICCS and FACS (31). The lungs were removed and pulmonary vasculature route. We have previously demonstrated that i.m. injection of ad- was perfused with 5 ml of warm buffer via the right ventricle of the heart. enoviral vector represents a convenient and efficient parenteral The lungs were cut into small pieces and subjected to 1-h collagenase (150 U/ml) digestion at 37°C and then mashed through a 70-␮m Falcon cell route of gene delivery (29). As a comparison, BCG was given via strainer with a 5-ml syringe plunger. Approximately 1 ϫ 107 splenocytes the conventional cutaneous route. Total splenocytes were isolated or lung-derived mononuclear cells were cultured in 5 ml of RPMI 1640 3 wk postimmunization and restimulated with different Ags ex medium in six-well plates with or without crude BCG (10 ␮l/well) and vivo. Mycobacterial Ag-stimulated IFN-␥ release was used as ␮ Ag85 (10 g/ml) Ag for 10 h, and were then treated with GolgiPlug (BD readout of type 1 immune activation. Marked elevated levels of Pharmingen, San Diego, CA) for an additional 5 h. Cells were harvested ␥ and washed with PBS and ICCS was conducted by using Cytofix/Cytoperm IFN- were readily measured in a dose-dependent manner in cells kits (BD Pharmingen) according to the manufacturer’s instructions. Briefly, 2 ϫ 106 cells were first stained with surface makers of FITC-CD8␣ or CyChrome CD8␣ or allophycocyanin-CD4 or FITC-CD4 in combina- tion with or without CyChrome-CD3, fixed, and permeabilized by Cytofix/ Downloaded from Cytoperm solution for 20 min at 4°C, and then labeled with PE-IFN-␥ Ab. A FACScan instrument was used (BD Biosciences, Sunnyvale, CA) to collect list mode data (200,000–500,000 total events) for analysis. Isolated splenocytes were stimulated with Ag85A-specific H-2d CD4 (LTSELPG WLQANRHVKPTGS) and CD8 (MPVGGQSSF) T cell peptides for5h(5 ␮g/ml) and processed as above for ICCS and FACS analysis. In some experiments, purified splenocytes were also subjected to an ELISPOT as- http://www.jimmunol.org/ say (31) to compare the frequencies of Ag-specific IFN-␥- and IL-13- releasing T cells. Pulmonary challenge with M.tb

Mice were challenged with live M.tb (H37Rv) by i.n. inhalation at the Level III facility in McMaster University or by aerosol inhalation at the Univer- sity of British Columbia. For i.n. delivery, 10,000 CFU of M.tb was diluted with PBS to a total volume of 25 ␮l and delivered into mouse lungs (14). For aerosol delivery, 100 M.tb bacilli per mouse were delivered via an inhalation exposure system (Glas-Col, Terre Haute, IN). The protective by guest on October 1, 2021 efficacy of vaccination with different immunization regimens was evalu- ated by plating serial 10-fold dilutions of lung and spleen homogenates in quadruplicates on Middlebrook 7H10 agar plates containing oleic acid- albumin-dextrose-catalase enrichment (Difco, Detroit, MI). Plates were in- cubated inside semisealed plastic bags at 37°C for 3 wk and colonies were counted. Cytokine measurement and histology The level of cytokines in the supernatants was measured by using mouse- specific ELISA kits (R&D Systems, Minneapolis, MN). The sensitivity of detection for IFN-␥, IL-4, and IL-13 was 2 pg/ml. For histology, lung tissues were fixed in 10% Formalin and processed for sectioning and H&E staining. Data analysis The difference comparison was conducted by using an unpaired, two-tailed Student t test. The difference was considered statistically significant when p Յ 0.05. Results Characterization of recombinant replication-deficient adenoviral vector expressing M.tb Ag85A (AdAg85A) Since secreted M.tb Ag has a stronger ability to elicit immune responses compared with nonsecreted Ags (32), AdAg85A was designed to encode a secreted form of Ag85A by replacing its endogenous signal peptide with human tissue plasminogen signal FIGURE 2. In vivo characterization of immune responses induced by AdAg85A. BALB/c mice were immunized i.m. with different doses of peptide (t-PA) sequence (Fig. 1a). Expression of Ag85A is under AdAg85A or 5 ϫ 107 PFU of Addl70-3 or s.c. with BCG and sacrificed 3 control of a murine CMV promoter. A strong mRNA signal with (a and b)or2(c) wk later. Splenocytes were isolated and cultured with or the predicted size was detected in the total RNA isolated from without Ag85 proteins or M.tb CFP. The level of IFN-␥ (a) or IL-13 (b)in A549 cells infected with AdAg85A but not with control vector culture supernatants was measured by ELISA. The number of IFN-␥-or Addl70-3 (Fig. 1b). Furthermore, using a mAb specific for Ag85A IL-13-releasing cells was determined by ELISPOT assay. Data represent and Western blotting, an Ag85A protein band of ϳ30 kDa was the mean value Ϯ SEM of triplicate samples. 6360 VIRAL-MEDIATED MUCOSAL IMMUNITY AGAINST TB from AdAg85A-immunized, but not Addl70-3-treated, mice follow- Distinct distribution of cellular immune responses following ing stimulation with mycobacterial Ags, particularly Ag85 proteins parenteral and respiratory mucosal AdAg85A vaccination ␥ (Fig. 2a). Not only were Ag85-specific IFN- responses elicited by We next investigated the potential difference in the distribution of ϫ 7 AdAg85A at doses above 1 10 PFU much stronger than those immune responses elicited by i.m. parenteral and i.n. mucosal vac- elicited by BCG immunization (50–100 ng/ml vs 8.8 ng/ml), but the cinations by comparing type 1 immune responses in different or- level of responses stimulated by M.tb CFP was also comparable to gans (draining lymph nodes, spleen, and lung). Intramuscular im- that by BCG vaccination, indicating the potent immunogenicity of munization with AdAg85A elicited a potent IFN-␥ response in the AdAg85A vaccine in vivo. To verify the type 1 nature of immune popliteal lymph nodes draining the site of injection after 1 wk, activation by AdAg85A, type 2 cytokines IL-4 and IL-13 were mea- which then quickly declined to undetectable levels at weeks 2, 4, sured. Although there were no measurable levels of IL-4 for all cul- and 6 (Fig. 3a). In contrast, the responses in the spleen peaked ture conditions (data not shown), the overall magnitude of Ag85- around week 2 and then declined to levels that were maintained specific IL-13 responses was only ϳ1% of Ag85-specific IFN-␥ over weeks 4 and 6 (Fig. 3b). Such potent immune response in the responses (Fig. 2b), indicating that AdAg85A vaccination elicits a spleen triggered by i.m. immunization was found to be accounted typical type 1 cellular immunity in vivo. To further verify the Th1 for primarily by Ag85A-specific CD8 T cells as assessed by ICCS nature of immune activation caused by AdAg85A vaccination, we immunoassay, whereby the ratio of IFN-␥-releasing CD8 vs CD4 compared the relative frequencies of IFN-␥- and IL-13-releasing T T cells was ϳ8:1 (Fig. 3c). In comparison, i.n. mucosal - cells in the spleen. Indeed, after AdAg85A immunization, the major- zation led to much smaller immune responses both in the medias- ity of Ag85-specific splenocytes were found to produce Th1 cytokine tinal lymph nodes draining the respiratory tract and in the spleen

IFN-␥ but not Th2 cytokine IL-13 (Fig. 2c). (Fig. 3, a–c). We next examined the level of immune responses in Downloaded from http://www.jimmunol.org/ by guest on October 1, 2021

FIGURE 3. Immune activation in local draining lymph nodes (LN), spleen, and lung induced by i.m. parenteral or i.n. mucosal immunization with AdAg85A. Splenocytes (a) and -derived cells (b) were isolated from mice immunized i.m. or i.n. with AdAg85A at weeks 1, 2, 4, and 6 and cultured with or without stimulation by Ag85 complex proteins, and the level of IFN-␥ was measured by ELISA. Data represent the mean value Ϯ SEM of triplicate samples. In separate experiments, splenocytes were isolated 2 wk after i.n. or i.m. immunization with AdAg85A and stimulated with CD4 T cell- or CD8 T cell-specific peptides and ICCS was conducted following surface immunostaining (c). The percentage in the lower right and upper left quadrants represents the frequency of Ag85A-specific IFN-␥-releasing CD4 and CD8 T cells out of the total cells, respectively. Total lung mononuclear cells were isolated at weeks 2 (d)or6(e) postimmunization with PBS (naive), control vector Addl70-3 i.n., or AdAg85A i.n. or AdAg85A i.m. and stimulated with Ag85 proteins and M.tb CFP. The number of Ag85A-specific IFN-␥-releasing CD4 and CD8 T cells was examined by intracellular IFN-␥ staining and the results are expressed as the absolute number of IFN-␥-positive (IFN-␥ϩve) cells per lung per group per time. Results are expressed as the mean Ϯ SEM from four to six mice per vaccinated group per time and average value from two mice per control group per time. ND, Not statistically .p Ͻ 0.05 ,ء ;significantly different The Journal of Immunology 6361 the lung. Since in contrast to the lymph nodes and spleen, the lung parenteral and mucosal AdAg85A . At 4 wk after contains many cell types other than lymphocytes, ICCS was used i.m. AdAg85A vaccination, while M.tb infection in the lung to analyze mycobacterial Ag-specific CD4 and CD8 T cell re- was reduced by ϳ0.5 log, there was little reduction in the spleen sponses. At 2 wk postimmunization, mucosal immunization with (Fig. 4, a and b). By 12 wk, i.m. AdAg85A vaccination-medi- AdAg85A induced both CD4 and CD8 T cell responses as ated protection completely vanished. In stark contrast, i.n. illustrated by marked increases in the number of Ag-specific AdAg85A vaccination reduced M.tb infection in the lung by 1.3 ␥ IFN- -releasing T cells as compared with that in mice receiving and 1.5 log at weeks 4 and 12 postvaccination, respectively mucosal delivery of PBS or Addl70-3 (Fig. 3d). In comparison, (Fig. 4, a and b). The CFU in the spleen was also significantly parenteral immunization with AdAg85A failed to induce a CD4 T reduced at these times by mucosal vaccination. In keeping with cell response while it resulted in a CD8 T cells response compa- potent immune protection, there was much reduced lung histo- rable to the level of CD8 T cell responses induced by i.n. immu- pathology upon airway M.tb challenge in mice immunized i.n. nization (Fig. 3d). At 6 wk postimmunization, both Ag-specific with AdAg85A (Fig. 4c). In comparison, a much greater extent CD4 and CD8 T cell responses induced by mucosal vaccination remained at high levels in the lung (Fig. 3e). In comparison, of histopathology was observed in the lung of i.m. AdAg85A- although there was still little CD4 T cell response in the lung immunized hosts (Fig. 4c). Potent immune protection mediated induced by i.m AdAg85A immunization, CD8 T cell responses by mucosal AdAg85A vaccination is associated with higher lev- declined by ϳ75% at 6 wk (Fig. 3e). These results suggest that els of retention of Ag85A-specific CD4 and CD8 T cells in the parenteral and respiratory mucosal AdAg85A vaccinations give lung (Fig. 3, d and e). To investigate whether both CD4 and rise to differential immune activation profile and distribution, with CD8 T cells contributed to immune protection, i.n. AdAg85A- the former primarily inducing a robust systemic activation of CD8 vaccinated mice were depleted of either CD4 or CD8 T cells or Downloaded from T cells and the latter inducing the activation/accumulation of both both, before M.tb challenge, with anti-CD4 and CD8 Abs. Al- CD4 and CD8 T cells in the lung. though i.n. AdAg85A reduced M.tb infection in the lung by Ͼ1.6 log, depletion of either CD4 or CD8 T cells markedly Potent protection from pulmonary TB conferred by respiratory attenuated such protection and depletion of both subsets com- mucosal AdAg85A vaccination and contribution by CD4 and pletely abolished such protection in the lung (Fig. 4d) and http://www.jimmunol.org/ CD8 T cells spleen (data not shown). These results suggest that both CD4 Having characterized the immune responses, we compared im- and CD8 T cells participate in mucosal AdAg85A vaccination- mune protection from pulmonary M.tb challenge conferred by mediated protection. by guest on October 1, 2021

FIGURE 4. Sustained immune protection from pulmonary M.tb challenge by respiratory mucosal, but not parenteral, vaccination with AdAg85A. Immunized or nonimmunized (naive) mice were challenged via the airway at either 4 or 12 wk postimmunization and the level of M.tb infection was determined in the lung (a) and spleen (b) 4 wk postchallenge. Results are expressed as the mean Ϯ SEM from 7 to 10 mice/group. H&E staining of lung sections from mice at 12-wk postimmunization with s.c. BCG or i.m. or i.n. AdAg85A (c). In separate experiments, groups of mice were immunized i.n. with AdAg85A for 4 wk and challenged via the airway with M.tb. Two days before challenge, some groups of mice were injected weekly i.p. with anti-CD4, anti-CD8 T cell Abs, or both for a total of 4 wk. These and control mice were sacrificed at 4 wk after M.tb challenge and the level of M.tb infection was determined in the lung (d). Results are expressed as the mean Ϯ SEM from seven mice per group. 6362 VIRAL-MEDIATED MUCOSAL IMMUNITY AGAINST TB

Single respiratory mucosal AdAg85A vaccination confers Intramuscular priming by DNAAg85A and respiratory mucosal improved protection from M.tb challenge over that by cutaneous boosting by AdAg85A confer the most potent immune protection BCG vaccination We next investigated whether systemic (i.m.) DNAAg85A vaccine Given potent protection by single i.n. AdAg85A vaccination, we priming and respiratory mucosal (i.n.) AdAg85A boosting could then compared i.n. AdAg85A vaccination with conventional cuta- further improve protection. Groups of mice were immunized in neous BCG vaccination. To decide on the strain of BCG to be parallel as depicted in Fig. 5a. Repeated i.m. DNAAg85A immu- used, we first compared the level of protection mediated by using nizations reduced M.tb infection by ϳ0.9 log in the lung at 4 wk a 0.5 ϫ 106 CFU dose of Connaught and Pasteur BCG strains. postchallenge but such protection subsided both in the lung and Although s.c. vaccination with both Connaught and Pasteur BCG spleen by 8 wk postchallenge (Fig. 5, b–e). The same was true strains similarly reduced M.tb infection in the lung (1–1.2 log) and with the mice receiving i.m. DNAAg85A priming and systemic spleen (1.1–1.3 log), the Connaught strain was slightly more potent (i.m.) AdAg85A boosting (Fig. 5, b–e). In contrast, systemic prim- (data not shown). Thus, the Connaught BCG was used for all of the ing with two repeated DNAAg85A injections and respiratory mu- following M.tb challenge experiments. Based on our previous (14) cosal boosting once with AdAg85A provided the most potent pro- and current observations, s.c. BCG vaccination at 4 and 12 wk tection of all of the tested vaccines/regimens, including s.c. BCG consistently leads to 1–1.2 log reduction of infection in the lung. In vaccination both at 4 and 8 wk postchallenge (Fig. 5, b–e), and this comparison, i.n. AdAg85A vaccination leads to 1.3–1.7 log reduc- level of protection was also better than single i.n. AdAg85A vac- tion (Fig. 4, a and d), thus already suggesting that i.n. AdAg85A cination at 8 wk postchallenge (4.42 Ϯ 0.18 CFU/lung; 2.74 Ϯ vaccination leads to an improved immune protection over that by 0.16 CFU/spleen). Again, such further enhanced immune protec-

s.c. BCG. To further verify, mice were vaccinated or treated in tion was found to be associated with more increased numbers of Downloaded from parallel for 6 wk with a control adenoviral vector (Addl70-3), i.m. Ag-specific CD4 and CD8 T cells in the lung (data not shown). AdAg85A, i.n. AdAg85A, or s.c. BCG. After airway M.tb chal- These results suggest that systemic priming with DNAAg85A vac- lenge, they were sacrificed at 4, 6, and 8 wk for colony assay. cine and airway mucosal boosting with AdAg85A vaccine repre- Intranasal delivery of Addl70-3 marginally enhanced protection sent an effective way to achieve a robust immune protection only at 4 wk compared with naive mice (Table I). Intramuscular against pulmonary TB. AdAg85A provided limited protection. Again, s.c. BCG led to a http://www.jimmunol.org/ 0.9–1.3 log reduction of infection in the lung and a 0.5–0.8 log reduction in the spleen at weeks 4, 6, and 8 postchallenge (Table Discussion I). In comparison, single i.n. AdAg85A immunization conferred Although different recombinant TB vaccines have been described the greatest extent of protection. At weeks 4, 6, and 8 postchal- in the past decade (9, 10, 15, 16, 32–40), the exploration of ad- lenge, the level of infection was reduced by 2.3, 2.1, and 1.5 log in enoviral vector for TB vaccination has not been reported. We have the lung and 2.4, 2.2, and 1.5 log in the spleen, respectively (Table now demonstrated that adenoviral-based recombinant TB vaccine, I). These results suggest that airway mucosal AdAg85A immuni- AdAg85A, elicits potent type 1 CD4 and CD8 T cell immune zation confers improved protection over s.c. BCG vaccination. We activation and single airway mucosal, but not i.m., immunization

have recently reported that compared with s.c. BCG vaccination, with AdAg85A provides potent protection against pulmonary M.tb by guest on October 1, 2021 respiratory mucosal BCG vaccination could also improve anti-TB challenge. Of importance, such protection is improved over that by protection, reducing M.tb infection by ϳ1.5 log and 1.4 log in the conventional cutaneous BCG vaccination at least for the periods of lung and spleen, respectively (14). Thus, respiratory mucosal time of observation. Airway mucosal AdAg85A immunization AdAg85A vaccination could accomplish a level of immune pro- confers an even further dramatically enhanced protection in the tection both in the lung and spleen that is comparable to or even mice that were systemically primed by DNAAg85A vaccine. To better than respiratory mucosal BCG vaccination. our knowledge, this is the first demonstration of superior immune

Table I. Comparison of immune protection by a single immunization with different vaccines/regimens at various times after airway M.tb Challenge a (log10 CFU/organ)

4wk 6wk 8wk

Lung Spleen Lung Spleen Lung Spleen

Naive 5.476 Ϯ 0.058 3.187 Ϯ 0.1065 5.717 Ϯ 0.125 3.756 Ϯ 0.186 5.702 Ϯ 0.110 4.192 Ϯ 0.128 (n ϭ 25) (n ϭ 17) (n ϭ 28)

Add170–3 (i.n.) 4.886 Ϯ 0.185¶ 2.536 Ϯ 0.351¶ 5.649 Ϯ 0.079 3.796 Ϯ 0.165 5.629 Ϯ 0.129 4.066 Ϯ 0.135 (n ϭ 7) (n ϭ 7) (n ϭ 11)

AdAg85A (i.m.) 4.991 Ϯ 0.299¶ 2.327 Ϯ 0.128§ 5.516 Ϯ 0.117 3.791 Ϯ 0.062 5.071 Ϯ 0.168¶ 3.248 Ϯ 0.159¶ (n ϭ 14) (n ϭ 11) (n ϭ 10)

AdAg85A (i.n.) 3.102 Ϯ 0.343†*** 0.706 Ϯ 0.255†**** 3.687 Ϯ 0.308†** 1.547 Ϯ 0.308†*** 4.249 Ϯ 0.121† 2.752 Ϯ 0.179†** (n ϭ 15) (n ϭ 16) (n ϭ 16)

BCG (s.c.) 4.610 Ϯ 0.078¶ 2.653 Ϯ 0.179§ 4.538 Ϯ 0.086† 2.946 Ϯ 0.142§ 4.371 Ϯ 0.114† 3.400 Ϯ 0.195§ (n ϭ 19) (n ϭ 13) (n ϭ 14)

a Mice were immunized with AdAg85A i.m. or i.n. or with BCG s.c. for 6 wk and were then challenged via the airway with M.tb. Naive mice or mice treated i.n. with a control adenoviral vector (Add170–3) were set up as control. A dose of 5 ϫ 107 PFU of adenoviral vector and a dose of 0.5 ϫ 106 CFU of Connaught BCG were administered. Mice were sacrificed at 4, 6, and 8 wk postchallenge. The number of tuberculous bacilli in the lung and spleen was determined by colony enumeration assay. Data are expressed as mean Ϯ SEM from pooled two to three separate experiments per time point per treatment. n, Number in parentheses represent the total number of mice per group per time p Ͻ 0.0001 as compared to BCG ,ءءءء ;p Ͻ 0.001 ,ءءء ;p Ͻ 0.005 ,ءء ;used for analysis. ¶, p Ͻ 0.05; §, p Ͻ 0.00001; †, p Ͻ 0.00000001 as compared to the naive group group (the difference is considered statistically significantly different when p Յ 0.05). The Journal of Immunology 6363 Downloaded from http://www.jimmunol.org/

FIGURE 5. Immune protection by heterologous prime-boost vaccination regimens. Schematic diagram of experimental design (a). Three repeated injections of DNAAg85A vaccine were conducted at a 3-wk interval. In some groups, AdAg85A was given i.n. or i.m. as the final booster. As control, some mice received only a single i.n. or i.m. injection of AdAg85A or s.c. BCG. Mice were challenged via the airway with M.tb at 10 wk after the last by guest on October 1, 2021 immunization and the level of infection in the lung and spleen was determined by colony enumeration assay at 4 wk (b and c)or8wk(d and e) postchallenge. Results are expressed as the mean Ϯ SEM from 8 to 12 mice/group per time.

protection conferred by the use of recombinant TB vaccine(s) ex- herpes vaccine. At the present time, it remains to be determined pressing a single M.tb Ag. Thus, adenoviral TB vaccine represents whether the quality of CD8 memory T cells in the lung generated a promising novel TB vaccine capable of potent mucosal immune by the two routes of AdAg85A immunization differs and what are protection. Our study also illustrates the critical advantage of the mechanisms by which mucosal AdAg85A immunization fa- respiratory mucosal vaccination over parenteral vaccination for vors memory T cell retention in the lung. Evidence suggests that ␣ ␤ protection against pulmonary TB. the interactions of 4 1 integrin/VCAM-1 and LFA-1/ICAM-1 ad- Respiratory mucosal immunization with AdAg85A results in a hesion molecules play a role in the airway tissue homing and re- preferential accumulation/retention of Ag-specific CD4 and CD8 T tention of memory T cells (44). Thus, it is very likely that the T cells in the lung but not in the distant lymphoid organs. This is in cells preferentially retained in the lung of airway mucosal-immu- contrast to parenteral AdAg85A vaccination that leads to the ac- nized hosts potently protect from pulmonary TB. This notion is cumulation of Ag-specific CD8 T cells predominantly in the further supported by our observation that depletion of both CD4 spleen. Of interest, regardless of a high number of such CD8 T and CD8 T cells abolishes immune protection and that further cells in the spleen of i.m. immunized mice, there was a relative enhanced protection by heterologous systemic prime/airway mu- lack of protection in the spleen. This could result from a greater cosal boost vaccination is closely associated with more increased systemic dissemination due to high levels of lung infection and numbers of Ag-specific T cells retained in the lung. perhaps also from a poorer protective property of these cells. Re- In the last decade or so, a number of new TB vaccines have been cently, strong evidence has emerged that memory T cells can be under development and these include recombinant plasmid DNA- localized not only to the secondary lymphoid organs but also to the and protein/peptide-based and viral-based vaccines (9, 10, 15, 16, peripheral tissues (41), and it is the memory T cells present in the 32, 38). Although nonorganism-based recombinant vaccines such lung that protect the host from secondary infection (42). Although as DNA and protein/peptide vaccines are believed to be safe, even parenteral AdAg85A immunization also leads memory CD8 T with repeated injections and/or adjuvant, none of those vaccine cells to be recruited into the lung, only mucosal vaccination results formulations confers the level of immune protection that exceeds in sustained accumulation/retention of both CD4 and CD8 T cells that by BCG vaccine in experimental models (in most studies in- in the lung. In this regard, Gallichan and Rosenthal (43) have cluding our current one, 0.3–1.3 and 0.3–1.0 log reduction of M.tb shown that long-term mucosal CTL memory was observed only infection in the lung and spleen, respectively, with DNA or subunit with mucosal, but not systemic, immunization with an adenoviral TB vaccination; compared with 0.5–1.4 log and 0.6–1.4 log in the 6364 VIRAL-MEDIATED MUCOSAL IMMUNITY AGAINST TB lung and spleen with BCG, respectively) (15, 16, 18). Moreover, primed hosts, it accomplishes a remarkable level of protection. The the limited accessibility to the pulmonary mucosal surface or low level of protection triggered by adenoviral-mediated mucosal vac- efficiency of gene transfer has restricted the airway mucosal use of cination could be even better than the current BCG vaccine given these TB vaccines. D’Souza et al. (18) have explored the use of parenterally or mucosally. Of note, BCG vaccination seems to hold lipid-encapsulated DNA TB vaccine for i.n. immunization but the off lung infection more constantly than AdAg85A vaccination. It is resultant immune protection is still unsatisfactory (0.7–1.0 and likely that activation of multiple T cell clones of different Ag spec- 0.1–0.3 log reduction in the lung and spleen, respectively). Re- ificity by BCG vaccination (AdAg85A produces only Ag85A-spe- combinant vaccinia has been used to express selected cific T cells) may confer an advantage of long-term lung protec- M.tb Ags but this type of viral TB vaccine by itself cannot elicit tion. We are currently investigating whether multivalent or strong immune protection. In comparison to all of these nonmy- mixture-based adenoviral TB vaccines expressing Ag85 and the cobacterial recombinant forms of TB vaccines, single airway mu- M.tb Ags such as ESAT-6, missing from BCG (48), could further cosal vaccination with our AdAg85A vaccine elicits an improved improve such immune protection. Our current findings also war- protection over cutaneous BCG vaccination (1.3–2.4 and 1.0–2.5 rant further evaluation in TB models involving the use of guinea log reduction of M.tb infection in the lung and spleen, respectively, pigs and other large animals. with i.n. AdAg85A compared with 0.9–1.3 and 0.5–1.3 log reduc- tion with s.c. BCG, respectively). Furthermore, this level of im- Acknowledgments mune protection is also comparable to or even better than that by We are grateful to Chuyan Ying, Duncan Chong, Xueya Feng, and airway mucosal BCG vaccination (1.5–1.7 and 1.0–1.5 log reduc- Kelly Dayball for their invaluable technical assistance. We also acknowl- tion in the lung and spleen with i.n. BCG, respectively) (14). edge the provision of M.tb genomic DNA, CFP, and Ag85 complex protein Downloaded from More recently, recombinant BCG- and auxotrophic M.tb-based by Colorado State University through the funds from the National Institute vaccines have also been evaluated. Compared with wild-type of Allergy and Infectious Diseases (Contract No1-AI-75320). BCG, recombinant BCG vaccine expressing ESAT-6 was found to confer a similar protection in mouse models while it conferred References better protection in the spleen, but not in the lung, of guinea pig 1. WHO vaccine preventable diseases: global summary. 2001. World Health Orga- models (34). Recombinant BCG overexpressing Ag85B appears to nization, Geneva, .

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